Folate cofactors are essential for the growth of all organisms. While man and animals are able to take folates from foods, most microorganisms have to synthesize folates de novo. Thus, enzymes involved in folate biosynthesis are targets of antimicrobial agents. 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the first reaction in the folate biosynthesis pathway. The long-term goals of the project is to elucidate the structure and mechanism of HPPK and to design inhibitors for the enzyme. HPPK was expressed in E. coli at high level. MALDI-MS analysis was used to determine the molecular weight of the recombinant HPPK. The result showed that the N-terminal methionine was removed in vivo.